Examination of the mechanism by which histone deacetylase inhibitors (HDI) effect prometaphase arrest in human tumor cell lines was continued. Focus was on the kinetochores, protein complexes that serve as attachment sites for microtubules on chromosomes. Immunofluorescence showed that treatment with HDI did not affect CENP-C, a resident centromeric protein, whereas staining of CENP-E, CENP-F and BUB-1 at the kinetochores was greatly reduced. The latter two proteins assemble on the kinetochore at prophase; in HDI-treated populations kinetochores of both prophase and prometaphase cells were bereft of CENP-F and BUB- 1. Western blots showed that the levels of these three proteins in HDI- treated cells were equal to or greater than those measured with nocodazole-treated populations of similar mitotic index. Treatment with HDI resulted in increased acetylation not only of histones but also of non-histone proteins in the human tumor cell lines; none of the three kinetochore proteins were acetylated following incubation of the cells with HDI. The most likely explaination for the HDI-induced prometaphase arrest is that the presence of hyperacetylated histones at the centromere interferes with kinetochore assembly. - mitosis, prometaphase arrest, kinetochores, histone deacetylase